Comparative structure activity and target exploration of 1,2-diphenylethynes in Haemonchus contortus and Caenorhabditis elegans

Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25, that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation.

Despite the significant burden of parasitic helminths on health, the majority of anthelmintics for use in humans were first developed as veterinary anthelmintics (Woods et al., 2007;Nixon et al., 2020).The challenges associated with anthelmintic discovery and development are numerous and often hindered by significant economic barriers (reviewed by Nixon et al., 2020).Since 2000, only two drugs, namely monepantel (Kaminsky et al., 2008) and derquantel (Lee et al., 2002), have been commercialised for use in livestock animals; neither of these compounds has yet been approved for use in humans.Despite the relatively slow rate of commercial drug development over the past decade, anthelmintic treatment remains a core component of helminth control programs.The reliance on anthelmintics to treat and control helminth infections has led to the widespread development of drug resistance in parasitic nematodes.Although difficult to establish for parasites of humans (Vercruysse et al., 2011), concerns about resistance have been raised in relation to parasitic nematodes such as Ascaris lumbricoides (see Krücken et al., 2017;Furtado et al., 2019) and Onchocerca volvulus (see Osei-Atweneboana et al., 2011).In parasitic nematodes of livestock animals, resistance is widespread and well-documented (reviewed by Kotze and Prichard, 2016;Kotze and Hunt, 2023).For example, the highly pathogenic, blood-feeding nematode of small ruminants, Haemonchus contortus and many related nematodes have become resistant in many countries to every class of anthelmintic currently on the marketexcept for derquantel, available as a combination therapy with abamectin (Startect®).However, there is evidence of reduced efficacy (93.8 % efficacy) of Startect® against H. contortus in Merino sheep (Sales and Love, 2016;Lamb et al., 2017), although it is unclear whether this finding is related to macrocyclic lactone and/or derquantel resistance development.Thus, the widespread development of anthelmintic resistance, coupled with a relatively slow rate of drug discovery, lends impetus to the development of new anthelmintics with novel mechanisms of action.
Some recent anthelmintic drug discovery efforts (reviewed by Herath et al., 2022) have been centred around two nematodesthe strongylid H. contortus and the free-living Caenorhabditis elegans.Both of these species represent useful models for anthelmintic drug discovery, because they can be readily maintained and produced in a laboratory setting and are both related to numerous socioeconomically important nematodes (clade V; order Strongylida) of animals and humans.Moreover, the extensive availability of genomic, transcriptomic and proteomic resources and tools for these two species (Wang et al., 2019;Doyle et al., 2020;Davis et al., 2022) provides a solid basis for detailed investigations of the modes and mechanisms of action of currently-available and novel anthelmintic compounds.In previous work, an established high-throughput, whole-organism, motility-based phenotypic screening assay of the "HitFinder" library (n = 14,400; Maybridge; cf.Taki et al., 2021a) identified a compound, HF-00014, that had significant anthelmintic activity against C. elegans.HF-00014, herein referred to as UMW-9729 (Fig. 1), was shown to inhibit the motility of young adults of C. elegans, displaying a half-maximal inhibitory concentration (IC 50 ) of 5.6 μM (88.9 % maximum motility inhibition).
Although the available background information on UMW-9729 was limited, it was proposed that there was significant potential for further pre-clinical anthelmintic development.UMW-9729 (Fig. 1) is composed of three aryl ring systems and an alkyne linker.Further, it was proposed that the synthesis of a UMW-9729 analogue series was feasible, with the relatively simple structure providing an opportunity to produce several bioactive compounds.As such, UMW-9729 presented as a promising candidate for further medicinal chemistry optimisation as a nematocide via a quantitative structure-activity relationship and a drug target identification study.Here, we (i) synthesised a series of UMW-9729 analogues; (ii) assessed the anthelmintic activity of these analogues on both H. contortus and C. elegans; (iii) evaluated the cytotoxicity and mitotoxicity of selected analogues on HepG2 human hepatoma cells; and (iv) inferred potential protein targets of UMW-9729 in each H. contortus and C. elegans.

H. contortus larvae and adult procurement
H. contortus (Haecon-5 strain; cf.Schwarz et al., 2013) was produced in experimental sheep as described previously (Schwarz et al., 2013;Preston et al., 2015) and in accordance with the institutional animal ethics guidelines (permit no.23983-2811-4; The University of Melbourne, Parkville, VIC, Australia).Helminth-free Merino sheep (six months of age; male) were orally inoculated with 7000 third-stage larvae (L3s) of H. contortus.Four weeks after inoculation, faecal samples were collected from sheep with patent H. contortus infection.These samples were incubated at 27 • C and >90 % relative humidity for one week to yield L3s (Preston et al., 2015), which were then collected in tap water and allowed to migrate through two layers of nylon mesh (pore size: 20 μm; Rowe Scientific, Doveton, VIC, Australia) to remove debris.Clean L3s were stored in the dark at 11 • C for up to six months (Preston et al., 2015).
Adult H. contortus were collected from the abomasa of sheep infected for 10 weeks, washed extensively with phosphate-buffered saline (PBS, pH 7.4) and subsequently in RPMI 1640 media supplemented with final concentrations of 2 mM L-glutamine, 100 IU/mL of penicillin, 100 μg/ mL of streptomycin and 0.25 μg/mL of amphotericin B (Thermo Fisher Scientific, Scoresby, VIC, Australia; this supplemented RPMI was designated RPMI*).Female and male worms were collected and separated immediately prior to compound testing.
The dose-response assay for H. contortus followed a well-established protocol (Taki et al., 2021b); it was employed to evaluate the potency of hit compounds against this nematode.Test compounds were assessed individually for an effect on the motility of xL3s (10-point, 2-fold serial dilution in LB*, 40 μM-0.16μM).Argentina).Over a period of 15 min, disturbance of an infrared beam in individual wells was recorded as an 'activity count'.Raw 'activity counts' for individual wells were normalised to the negative-controls.The compound concentrations were log 10 -transformed and fitted using a variable slope four-parameter equation, using the ordinary least squares fit model, employing Prism (v.9.1.0GraphPad Software, San Diego, CA, USA).Larval development was established at 168 h of incubation with compound, as described previously (Preston et al., 2015).The phenotypes of larvae were examined using a microscope (Preston et al., 2015) and recorded.

C. elegans preparation and dose-response assay
For the assay, C. elegans (N2 -wildtype Bristol strain) was maintained in the laboratory under standard conditions at 20 • C on nematode growth media (NGM) agar plates, with Escherichia coli OP50 as a food source (Stiernagle, 2006).Gravid adult worms were collected from NGM plates, washed with sterile M9 buffer and then treated with a solution containing 0.4 % (v/v) sodium hypochlorite and 170 mM sodium hydroxide for 4-8 min at 22-24 • C (room temperature) to release eggs (Stiernagle, 2006;Porta-de-la-Riva et al., 2012).The eggs were then washed five times with 15 mL of sterile M9 buffer (centrifugation at 500×g, 2 min).After washing, the egg pellet was suspended in 8 mL of M9 buffer in a 15 mL tube and gently agitated for 24 h at 22-24 • C to produce first-stage larvae (L1s); 45 h prior to screening, synchronised C. elegans L1s were inoculated on to NGM plates containing 500 μL of E. coli OP50 (~3000 larvae per plate) and allowed to develop to fourth-stage larvae (L4s) at 20 • C. L4s were collected from plates and washed twice with sterile M9 buffer by centrifugation (500×g, 2 min) to remove E. coli OP50, and then resuspended to a concentration of 100 larvae per 50 μL in sterile (autoclaved) LB*.
The dose-response assay for C. elegans followed a well-established protocol (Taki et al., 2021a) and was employed to evaluate the potency of hit compounds against this nematode.Test compounds were assessed individually for an effect on the motility of C. elegans (10-point, 2-fold serial dilution in LB*; from 40 μM to 0.16 μM) in the transition from the L4 to the young adult stage.Two compounds, monepantel (Zolvix™; Elanco, Australia) and moxidectin (Cydectin®; Virbac, France) were used as positive controls and prepared in the same manner as the test compounds.A solution of LB* +0.25 % (v/v) dimethylsulfoxide (DMSO) was used as a negative control.The test compounds and positive control compounds were arrayed in triplicate across individual flat-bottom 96-well microplates (cat.no.3596; Corning, Corning, NY, USA), with six wells on each plate containing the negative control.
Added to each well were 100 C. elegans in 50 μL of LB* to give a final volume of 100 μL.Plates were then placed in an incubator (Heratherm, model no.IMP180, Thermo Fisher Scientific, USA) at 20 • C for 40 h.At 40 h, worm activity (i.e.motility) was measured using a WMicroTracker ONE unit (Phylumtech, Sunchales, Santa Fe, Argentina).Over 15 min, disturbance of an infrared beam in individual wells was recorded as an 'activity count'.Raw 'activity counts' for each well were normalised to the negative controls.The compound concentrations were log 10 -transformed and fitted using a variable slope four-parameter equation, using the ordinary least squares fit model, employing the program Prism (v.9.1.0GraphPad Software, San Diego, CA, USA).

Assessment of the activity of selected compounds on H. contortus adults
The activity of UMW-9729 and six derivatives (12, 14, 15, 16, 18 and 25) was assessed on adult female specimens of H. contortus in an established assay (Taki et al., 2020).The compound was added in triplicate to the wells of a 24-well plate (cat.no.3524; Corning, USA) at a concentration of 40 μM in 500 μL of RPMI* (RPMI supplemented with final concentrations of 2 mM L-glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B; Thermo Fisher Scientific, Scoresby, VIC, Australia).Two positive-control compounds, monepantel and moxidectin, and a negative control containing 1 % (v/v) DMSO only, were included in triplicates on the same plate.Three adult females were added to each of the triplicate wells containing either the test compound or the controls and placed in a CO 2 incubator (10 % (v/v) CO 2 , 40 • C, >90 % relative humidity) for 1 day.A video recording (30 s) of each well was taken at 3 h, 6 h, 12 h and 24 h during the total incubation period to assess the reduction in worm motility, which was scored as 3 ("good"), 2 ("low"), 1 ("very low") or 0 ("no movement"; cf.Taki et al., 2020).For each test or control compound, the motility scores for each of the triplicate wells were calculated, normalised with reference to the negative control (100 % motility) and recorded as a percentage.
Monepantel and moxidectin (prepared in the same manner as the test compounds) were included as reference compounds.M-666 (10 M; Le et al., 2018) was used as a positive control; 0.25 % DMSO was used as a negative control.HepG2 cells were seeded into wells of a 96-well plate in 80 μL of DMEM* (at 1 × 10 5 cells per well) and allowed to adhere for 16 h (5 % (v/v) CO 2 , 37 • C, >90% humidity)prior to incubation with individual compounds, at a final volume of 100 μL.Cells were starved of serum (DMEM* without FBS) for 4 h prior to the incubation with compounds (Swiss and Will, 2011;Kamalian et al., 2015).Following 48 h of incubation with compounds, cell viability was determined by crystal violet staining ( Śliwka et al., 2016).The absorbance (595 nm) of treated cells was normalised using the negative-controls to calculate the cell viability.All compounds and controls were tested in triplicate.To determine the half-maximal mitotoxic concentration (MC 50 ) values, compound concentrations were log 10 -transformed, baseline-corrected using M-666, and fitted using a nonlinear regression four-parameter fit analysis using Prism v.9.1.0.
2. 1.6.1. Preparation of protein extracts from H. contortus and C. elegans. H. contortus (2,000,000 L3s) and C. elegans (500,000 young adults) were prepared as previously described, concentrated (separately) by centrifugation (2000×g, 5 min) and frozen at − 80 • C, following the removal of the supernatant.Subsequently, the frozen pellets were ground to a fine powder in liquid nitrogen using a mortar and pestle, each transferred to an individual 10 mL tube, suspended in 3 mL ice-cold phosphate-buffered saline (pH 7.0) containing 0.5 % (v/v) nonyl phenoxypolyethoxylethanol (NP-40) and lysed by gentle aspiration/expulsion using a 5 mL sterile syringe with a 22-gauge needle.Subsequently, the supernatant was collected from each suspension following centrifugation at 20,000×g for 20 min at 4 • C. The protein concentration in both supernatants were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA), adjusted to 2 mg/mL, and both supernatants were divided into four 250 μL aliquots (each containing 500 μg protein).
2.1.6.3.In-solution digestion and isobaric stable isotope labelling of peptides.Proteins in aliquots (45 μL) of individual samples (n = 80) were denatured in 8 M urea for 30 min at 37 • C and diluted to < 2 M urea using lysis buffer prior to processing for in-solution digestion (Ang et al., 2011).Samples were reduced with 10 mM Tris (2-carboxyethyl) phosphine, alkylated with 55 mM iodoacetamide, followed by digestion with trypsin (Promega) at 37 • C for 16 h.The trypsin-treated samples were acidified with 1.0 % (v/v) formic acid (FA) and purified using Oasis HLB cartridges (Waters, USA); wash solvent, 0.1 % FA; elution solvent, 80 % acetonitrile (CH 3 CN) in 0.1% FA).Then, proteins were labelled with tandem mass tags (TMTs) (Zecha et al., 2019).In brief, desalted peptides were resuspended in 50 mM triethylammonium bicarbonate (pH 8.5) and labelled with isobaric compounds using TMT10plex isobaric label reagent (Thermo Fisher Scientific, USA) that was dissolved in 41 μL of anhydrous CH 3 CN.The TMT-peptide mixture was incubated for 1 h at 25 • C with gentle shaking.Subsequently, 3.2 μL of 5 % (w/v) hydroxylamine was added to the mixture and incubated for 15 min at 25 • C with gentle shaking to quench the reaction.Labelled peptides were combined accordingly and then desalted on Oasis HLB cartridges (using wash solvent, 0.1 % FA; elution solvent, 80 % CH 3 CN in 0.1 % FA).Each mixed peptide sample was separated into eight fractions using the high pH reversed-phase peptide fractionation kit (Pierce), according to the manufacturer's protocol.All fractions were freeze-dried prior to resuspension in aqueous 2 % (w/v) CH 3 CN and 0.05 % (w/v) trifluoroacetic acid (TFA) before LC-MS/MS analysis.
Mass spectrometry data were processed using MaxQuant (v2.1.1.0)for the identification and quantification of peptides/proteins.Proteins were matched to those inferred from the reference genome (version 4) for H. contortus (Doyle et al., 2020) or C. elegans (PRJNA13758).The TMT reagent was corrected for natural carbon isotopes and incomplete stable isotope incorporation.Fixed modifications of carbamidomethylation of cysteine.Trypsin/P was set as the protease with a maximum of 2 missed cleavages.Variable modifications are oxidation of methionine and acetylation of protein N-terminus.All quantitative values were normalised based on the weighted ratio to reference channel function to the 1st TMT reference channel (126C) made up of a pool of each sample.The isobaric matching between runs feature to improve reporter ion-based quantitation was also turned on.Protein and PSM false discovery rates (FDR) were both set at < 0.01.Results are available via the PRIDE data repository (accession number: PXD048945).
2.1.6.5.Data processing and analysis.The quantitative protein data produced by MaxQuant was taken for analysis in R (v4.1.2).Decoy proteins, contaminant proteins, proteins only identified by modified peptides, and proteins that were identified by less than 2 razor or unique peptides were removed.Corrected reporter ion intensities were then divided by the intensity of the 37 • C channel.Due to the marked decrease in overall protein abundance with increasing temperature, protein abundance ratios were grouped by treatment temperature and subjected to quantile normalisation using the software package limma (v3.50.0;Ritchie et al., 2015).Proteins were filtered to retain only those with non-zero values for each sample, and these were taken for subsequent analysis.
Thermal profiles of quantified proteins were assessed using the package NPARC (v1.6.0;Childs et al., 2019), which fits nonparametric models to the temperature profile data under null and alternative hypotheses; p-values were then calculated from F-statistics with empirically estimated degrees of freedom, as described in the NPARC package documentation (Perrin et al., 2020).Melting profiles were plotted and manually inspected for top ranking protein hits that were statistically significant (Benjamini-Hochberg-adjusted p-values were <0.01).

General chemistry experimental
All non-aqueous reactions were performed under an atmosphere of nitrogen, unless otherwise specified.Commercially available reagents were used without further purification.Flash chromatography was performed with silica gel 60 (particle size 0.040-0.063μm) on a Com-biFlash Rf Purification System (Teledyne Isco) with mobile phase gradients as specified.NMR spectra were recorded on a Bruker Avance DRX 300 with the solvents indicated ( q H NMR at 300 MHz).Chemical shifts are reported in ppm on the δ scale and referenced to the appropriate solvent peak.Chemical shifts reported in 19 F NMR are referenced to an external standard (trifluoroacetic acid) in the solvent indicated (Rosenau et al., 2018).LCMS were analysed on an Agilent LCMS system H.T. Shanley et al.

Synthesis of analogues
To develop a structure-activity profile, a series of structural changes were made to define the anthelmintic activity of UMW-9729.The compound series was then assessed for potency in a dose-response assay on exsheathed third-stage larvae (xL3s) of H. contortus and young adults of C. elegans.
At the 3-position of the aryl ring (Table 2), incorporation of a -Cl (15) or -OCF 3 (13) group resulted in a complete loss of activity (>40 μM), whereas a 3-OH derivative (17) showed greatly reduced activity (29 μM, MMI of 54 %).The addition of a -Me group (11) displayed slightly lower activity (13 μM), whereas incorporation of -CF 3 (12) or -F ( 14) retained activity equipotent activity (8.2 and 5.2 μM respectively; Additional File 1: Fig. S1), relative to UMW-9729.This retention of the potency of both 12 and 14, compared to the loss in activity in 13 and 15, indicated that small, electron-withdrawing groups may be preferred at the 3 position.Finally, potency assessment of a 3-pyridine derivative ( 16) was found to be equipotent in H. contortuspossibly providing a pathway for future inclusion of polar groups (Additional File 1: Fig. S1).

Changes to alkyne linker resulted in a loss of activity
Changing the point of attachment of the phenylacetylene motif (26) (Table 5) resulted in a complete loss of activitysimilarly, reduction of the alkyne linker (Table 5) to its alkyl counterpart (28, IC 50 = 16 μM, MMI of 57 %) or replacement with an oxadiazole functional group (33, IC 50 = 2.5 μM, MMI of 55 %) resulted in a significant reduction of activity.Finally, replacement of the terminal phenyl with a trimethyl silyl group (23), or removal of the terminal phenyl ring (27), also resulted in a complete loss of activity.Attempts to incorporate a pyridine moiety within the central phenyl ring (24) also reduced activity (IC 50 = 24 μM, MMI of 70 %).

Substituent addition to terminal phenyl ring overall linked to activity loss
The panel of compounds which were tested for activity on larvae of H. contortus were also tested for inhibitory effects on the motility of young adults of C. elegans.Here, UMW-9729 displayed an IC 50 of 14 μM, reaching an MMI of 85 % (Additional File 1: Fig. S2).Comparatively, monepantel and moxidectin displayed IC 50 values of 0.03 μM (MMI = 93 %) and 0.003 μM (MMI = 100 %), respectively.
Changes to the 3-position of the terminal phenyl ring (Table 2) were generally well-tolerated compared to the 2-position.Incorporation of   a Dash '-'indicates that X = 'C'.b IC 50 calculated from three independent assays in triplicate.c (Maximum motility inhibition, %).  3) all led to a loss of motility inhibition in C. elegans (>40 μM).

Replacement of alkyne and removal of terminal phenyl loses activity
Changing the phenyl acetylene point of attachment ( 26), removal of the terminal phenyl ring ( 27), reduction of the alkyne (28) or replacement of the alkyne with an oxadiazole motif (33) resulted in a loss of activity (>40 μM), indicating that the rigidity of UMW-9729 provided by the alkyne group is pivotal to the compound's nematocidal activity in C. elegans.Incorporation of a nitrogen within the central phenyl ring (24) caused a loss in activity.

Proteomic investigation to infer targets in H. contortus and C. elegans
To investigate the possible protein targets of UMW-9729 in the nematode models, protein lysates of xL3s of H. contortus and L4s of C. elegans were individually incubated with 50 μM of UMW-9729 a then subjected to TPP across a gradient of 37  S3).Statistically significant protein target candidates (Benjamini-Hochberg adjusted p-values (pAdj) < 0.01) were then plotted and manually inspected; three proteins, designated HCON_00134350, HCON_00021470 and HCON_00099760, were stabilised in the presence of UMW-9729 (Additional File 1: Table S1).

Discussion
Here, we demonstrated that UMW-9729 displayed moderate anthelmintic activity against larvae of the highly pathogenic model nematode, H. contortus, and identified two derivatives, 18 and 25, with a 3-fold increased activity when compared to the parent molecule.Further, we highlighted some key structural features contributing to the inhibition of worm motility.In brief, we found that, with the exception of an ortho -F or a para -Me functional group, additions to the terminal phenyl ring at either the ortho or para position were not tolerated.At the meta position, -Me, -CF 3 and -F additions resulted in equipotent derivativeshowever, inclusion of a bulkier -OCF 3 or -Cl group resulted in a loss of activity.These differences suggest that the terminal phenyl may be oriented towards a binding cavity to accommodate the terminal aryl ring; however, the increased activity shown for the para -Me compound 18 suggests that there is space to extend further into this pocket.Moreover, N-methylation of the pyrazole motif on the UMW-9729 scaffold identified a compound (25) with greater activity, whereas a scaffold-hop to an oxadiazole moiety retained potency.Finally, efforts to replace the alkyne linker with a less rigid alkyl group, or an oxadiazole isostere, were not favourable towards activity.Of note, although no analogue synthesised here was more potent than monepantel, several analogues, including UMW-9729, were ~3-fold more active than moxidectin.Additionally, the activity of moxidectin was relatively moderate, considering the reported anthelmintic in vivo field efficacy.We also tested UMW-9729 and several key derivatives (12, 14, 15, 16, 18 and 25) for nematocidal effects against adult females of H. contortus; however, only one compound, 25, displayed a moderate motility inhibition after 24 h of incubation.Thus, the apparent low effect of UMW-9729 against this parasite's most pathogenic stage (i.e.adult) of this parasite does suggest that the development of this compound as an effective anthelmintic may be limited.
The activities of UMW-9729 and its derivatives were also explored against C. elegans.Although we validated UMW-9729 as a moderate inhibitor of C. elegans motility, in general, structural changes were not well tolerated.On the terminal phenyl ring of UMW-9729, substitutions at the ortho or para position lost activity against this worm species, whereas only electron-withdrawing groups (such as -F, -Cl or -CF 3 ) at the meta position demonstrated equipotent activity.Moreover, N-methylation of the pyrazole moiety gave a loss of activity, yet an oxadiazole motif was equipotent.Finally, changes to the alkyne linker, removal of the terminal phenyl ring or replacement with an oxadiazole motif also resulted in a loss of activity.Moreover, all analogues tested, including UMW-9729, were substantially less active against C. elegans than monepantel or moxidectin.
Several key differences in the activity of analogues between both H. contortus and C. elegans suggest that UMW-9729 may target two or more structurally distinct proteins in both nematode species.For  instance, although the para-methyl derivative 18 was 3-fold more active than UMW-9729 in H. contortus, interestingly, this derivative was inactive against C. elegans.Another N-methylated pyrazole derivative, 25, was also inactive against C. elegans, yet 3-fold more active against H. contortus.Conversely, a meta-Cl substitution on the terminal phenyl ring produced an analogue (15) with ~ 2-fold increased activity than UMW-9729 against the free-living nematode species, contrasting a loss of activity against the parasitic worm.These differences in activity might be explained by the biological differences between C. elegans (free-living) and H. contortus (parasitic).In a future study, it would be of interest to assess the present collection of UMW-9729 analogues against other, closely related parasitic nematode species to identify whether there is a shared pharmacophore among these parasitic organisms.
To understand the mechanism of action responsible for the anthelmintic activity of UMW-9729, we used TPP (Savitski et al., 2014;Mateus et al., 2020;Taki et al., 2022) to identify UMW-9729-bound proteins in a lysate of H. contortus larvae.Here, we identified and prioritised three H. contortus proteins (named HCON_00134350, HCON_00021470 and HCON_00099760) which were significantly stabilised in the presence of UMW-9729.In each case, the function of the protein was inferred from the primary amino acid sequence (Doyle et al., 2020) and from the related C. elegans orthologue; in short, HCON_00134350 (C.elegans orthologue GLB-1, 54.9 % sequence identity, E-value of 6.8 × 10 − 51 ; Tilleman et al., 2011) was predicted to be a globin domain-containing protein whose function is associated with heme-binding; HCON_00021470 (C.elegans orthologue CDC-5L, 81.2% identity, E-value of 0; Shiimori et al., 2013) was predicted to be a cell division cycle 5-like protein whose function is associated with mRNA splicing; HCON_00099760 (C.elegans orthologue LPR-2, 66.7 % identity, E-value of 1.3 × 10 − 114 ; Forman-Rubinsky et al., 2017) was predicted to be apolipoprotein D whose function is linked to retinoid binding activity.Although the functions of the H. contortus proteins identified here have not yet been fully established, it is possible that the disruption of one or more of these proteins leads to worm immobilisation.
Notably, none of the proteins predicted here as targets of UMW-9729 were shared by C. elegans and H. contortus.In concert with the SAR investigation, this finding suggests that UMW-9729 does not share a protein target in both species.It is possible that the functional processes altered/interrupted by UMW-9729 in the parasitic nematode are not present in the free-living C. elegans worm (cf.Geary and Thompson, 2001) and, hence, UMW-9729 achieves anthelmintic activity via divergent pathways.Although these results may question the use of C. elegans as a surrogate model for antiparasitic discovery, it is clear, through the development of monepantel (Kaminsky et al., 2008) and the anthelmintic candidate Nemacol (Harrington et al., 2023), that C. elegans remains a useful system, with the caveat that drug testing also needs to be undertaken against one or more pertinent parasitic nematodes, including H. contortus.
An alternative explanation is that, in the case of UMW-9729, TPP may not be adequate to unequivocally define the target(s) of this compound.Possibly, orthogonal approaches, aimed at validating the protein targets identified here, could illuminate the genuine mode(s) of action of UMW-9729 in a nematode model.Complementary protein-focussed investigations, such as isothermal dose-response fingerprinting (Jafari et al., 2014) or affinity-based assays (Him et al., 2009;Seo and Corson, 2019), or genomics-directed studies, such as RNA interference (Blanchard et al., 2018;Hou et al., 2023), CRISPR/Cas9 genome editing (cf.Waaijers et al., 2013;Quinzo et al., 2022) or resistance-based studies utilising either H. contortus (see Kaminsky et al., 2008) or C. elegans (see Burns et al., 2006), could be employed to identify and/or validate drug-protein interactions.Of note, the binding mode of a structurally similar aryl alkyne compound, designated CHIR-090, in complex with a gram-negative bacteria specific protein has been previously elucidated (Brown et al., 2012).Although (presumably) CHIR-090 and UMW-9729 do not share the same target, the binding pocket interactions could be similar given the shared 1,2-diphenylethyne chemical moiety, and the interactions identified there (Brown et al., 2012) could assist future mechanism of action studies.Similarly, in silico methods (Trott and Olson, 2010) could also be used to generate hypotheses as to how UMW-9729 and its derivatives interact with identified proteins, to understand whether predicted interactions are reflected in the SAR results.
Given the divergence in the nematocidal pharmacophore between H. contortus and C. elegans worms, the low-to-moderate activity against H. contortus adult worms, and the apparent lack of a conserved nematode drug target, the future development of UMW-9729 as a broadspectrum anthelmintic may be challenging.Certainly, future work should focus on the development of a non-cytotoxic and non-mitotoxic compound (with adequate pharmacokinetic properties) which is active against parasitic stages of H. contortus and other socioeconomically important nematodes (Keiser et al., 2016;Keiser and Häberli, 2021).Moreover, if a UMW-9729 analogue were established as a suitable front-runner candidate in vitro, it would be pivotal to also assess its antiparasitic activity in vivo.Finally, the validation of the protein targets inferred here, through complementary and/or orthogonal approaches, would be critical for the development of a UMW-9729-derived anthelmintic compound with a novel mechanism of action.
Fig.S2) -in contrast, a -Me derivative (11) lost activity, suggesting that electron-withdrawing, lipophilic functional groups are preferred at this position.Finally, the incorporation of a 3-pyridine motif (16) resulted in a loss of activity; however, the inclusion of a 3-hydroxy (17) only slightly reduced activity (25 μM IC 50 ), suggesting that the incorporation of hydrophilic functional groups at the 3-position may be possible.Changes at the 4-position (-Me (18), -OMe (19), -Cl (20), -CN (21), and 4-pyridine (22; Table3) all led to a loss of motility inhibition in identify proteins which are stabilised in the presence of UMW-9729.Using this technique, we first identified and quantified 4122 H. contortus proteins.Utilising a non-parametric analysis of the response curves(NPARC v.1.6.0;Childs et al., 2019), we assessed the thermal profiles of individual proteins and yielded 3270 melting profiles (Additional File 2: Table calculated from three independent assays in triplicate.b (Maximum motility inhibition, %).

Fig. 2 .
Fig. 2.The in vitro motility inhibition (%) of UMW-9729 and six active derivative compounds (12, 14, 15, 16, 18 and 25) against adult females of Haemonchus contortus, with reference to two control compounds (monepantel and moxidectin).Motility scores (assessed at 3-, 6-, 12-and 24-h time points) for each compound were calculated and normalised to a negative control (100 % motility), and were recorded as a percentage.Data points represent one experiment conducted in triplicate; the mean ± standard deviation (SD).

Table 2
Activity of 3-substituted benzene UMW-9729 analogues on larvae of H.